Search

Aims: Bisphenol A (BPA) is an endocrine-disrupting chemical inducing several damages such as neurotoxicity, immunotoxicity, and metabolic disorders. Obesity is the main risk factor for the increased occurrence of metabolic alterations as well as mood disorders. Here, we investigated in obese mice the effects of BPA on anxiety-like behavior, associated with neuroinflammation and immune activation. Main methods: Male C57Bl/6J mice were divided into 4 groups: control group (STD) receiving chow diet and BPA vehicle; STD group treated with BPA (50 μg/kg/die); high-fat diet (HFD) group receiving BPA vehicle; HFD group treated with BPA. BPA treatment started 12 weeks after HFD feeding and lasted 3 weeks. Key findings: The open field and elevated plus-maze tests showed in HFD + BPA group the worsening of HFD-induced anxiety-like behavior. The anxiogenic effects of BPA also emerged from hyperactivation of the hypothalamus-pituitary-adrenal gland axis, determined by the increased transcription of Crh and its receptor in the prefrontal cortex (PFC). Furthermore, BPA activated NLRP3 inflammasome and exacerbated the neuroinflammation induced by HFD, increasing IL-1β, TNF-α and monocyte chemoattractant protein (MCP)-1 in PFC. Furthermore, it induced inflammation and monocyte recruitment in hypothalamus and amygdala. Contextually, BPA significantly amplified the immune activation caused by lipid overload as evidenced by the increased expression of TLR-4 and MCP-1 in the PFC and triggered mastocytosis in the hypothalamus rather than STD mice. Significance: All these data show that sub-chronic BPA exposure represents an additional risk factor for mood disorders strictly related to obesity, enhancing neuroinflammation and immune activation triggered by HFD feeding.

Food is usually the major source of human exposure to environmental contaminants like heavy metals and synthetic compounds. This study proposes a quick and simple approach to combine the estimate of the intake of certain pollutants with the diet, in combination with different nutritional plans (Mediterranean diet, weight loss and for athletes). The estimation of the intake of three heavy metals and two perfluoroalkyl substances was carried out by entering the type and quantity of the foods provided by each of the three selected dietary plans in the UltraBio® app. Recurring elements are high levels of Cd and Pb and very low levels of PFASs, for all the plans considered. The Mediterranean diet scheme was the one with the lowest intake of all contaminants, which, in any case, remains within the safety limits by a large margin. The high protein diet leads to exceeding the limits for two metals and critical values for the third. The advantages of this approach are mainly represented by the possibility of having a personalized risk assessment of the intake of important food contaminants for the prevention of exposures that, over time, could put health at risk.

Endocrine-disrupting chemicals (EDCs) are agents able to exert perturbation toward the endocrine system via a broad array of signalling pathways. Some EDCs are released into the environment as a result of antropogenic activities. Analytical surveillance plays a critical role in investigating the prevalence of such chemicals in environmental samples. A study was carried out in a lagoon in Southern Italy, a water basin relates to the sea through a mouth channel, making this water body a "dynamic environment". The screening of fourteen EDCs in surface waters and sediments, includes a fast and cost-effective sample preparation, based on a solid-liquid (sediments) and liquid-liquid (surface waters) extraction and a chromatographic analysis by liquid chromatography tandem UV and fluorescence detection. Only four chemicals out the fourteen investigated EDCs were detected in both matrices with a frequency higher than 60%. The average concentrations of the single EDC were higher in sediments (730-155.000 ng kg-1 dw) than in surface waters (132-28.000 ng L-1 ). Limited to the assayed EDCs, the ecosystem has a low risk regarding to the conservation of biodiversity of the animal species living thereby, since the total estrogenic activity does not exceed 1 ng L-1 . PRACTITIONER POINTS: Occurrence of selected EDCs was investigated in an Italian lagoon in Southern Italy. BPAF, BADGE, and BPA were the most frequently and highly detected compounds in both waters and sediments. Concentration levels were greater in the sediment than in water samples. Low risk for the ecosystem biodiversity concerning investigated EDCs.

Polybrominated diphenyl ethers (PBDEs) are a class of flame-retardant organohalogen pollutants that act as endocrine/neuroendocrine disrupting chemicals (EDCs). In humans, exposure to brominated flame retardants (BFR) or other environmentally persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) and novel organophosphate flame retardants has been associated with increasing trends of diabetes and metabolic disease. However, the effects of PBDEs on metabolic processes and their associated sex-dependent features are poorly understood. The metabolic-disrupting effects of perinatal exposure to industrial penta-PBDE mixture, DE-71, on male and female progeny of C57BL/6N mouse dams were examined in adulthood. Dams were exposed to environmentally relevant doses of PBDEs daily for 10 weeks (p.o.): 0.1 (L-DE-71) and 0.4 mg/kg/d (H-DE-71) and offspring parameters were compared to corn oil vehicle controls (VEH/CON). The following lipid metabolism indices were measured: plasma cholesterol, triglycerides, adiponectin, leptin, and liver lipids. L-DE-71 female offspring were particularly affected, showing hypercholesterolemia, elevated liver lipids and fasting plasma leptin as compared to same-sex VEH/CON, while L- and H-DE-71 male F1 only showed reduced plasma adiponectin. Using the quantitative Folch method, we found that mean liver lipid content was significantly elevated in L-DE-71 female offspring compared to controls. Oil Red O staining revealed fatty liver in female offspring and dams. General measures of adiposity, body weight, white and brown adipose tissue (BAT), and lean and fat mass were weighed or measured using EchoMRI. DE-71 did not produce abnormal adiposity, but decreased BAT depots in L-DE-71 females and males relative to same-sex VEH/CON. To begin to address potential central mechanisms of deregulated lipid metabolism, we used RT-qPCR to quantitate expression of hypothalamic genes in energy-regulating circuits that control lipid homeostasis. Both doses of DE-71 sex-dependently downregulated hypothalamic expression of Lepr, Stat3, Mc4r, Agrp, Gshr in female offspring while H-DE-71 downregulated Npy in exposed females relative to VEH/CON. In contrast, exposed male offspring displayed upregulated Stat3 and Mc4r. Intestinal barrier integrity was measured using FITC-dextran since it can lead to systemic inflammation that leads to liver damage and metabolic disease, but was not affected by DE-71 exposure. These findings indicate that maternal transfer of PBDEs disproportionately endangers female offspring to lipid metabolic reprogramming that may exaggerate risk for adult metabolic disease.

Every day, people use personal care products containing UV filters. Although their use initially showed a protective role, toxicity is a concern for human health as several UV filters are endocrine-disrupting chemicals (EDCs). Exposure to EDCs may induce cardiovascular diseases and can affect the health of sensitive people, such as pregnant women. Currently, the world’s most widely used UV-B filter is octylmethoxycinnamate (OMC), an EDC. However, the disruptive effects on pregnant women are little known. The present work proposed to understand how long-term exposure to OMC affects vascular homeostasis. Endothelium-denuded human umbilical artery (HUA) rings were incubated in an organ bath system. Long-term effects of exposure to OMC (0.001–50 μmol/L) were evaluated on the contractile responses of HUA to the application of the contractile agents, serotonin (5-HT) and histamine (Hist). To investigate in more detail the vascular mode of action of OMC, through which it impairs the vascular homeostasis of HUA, the activity and expression of different 5-HT and Hist-receptors involved in contractility processes were studied. Our findings pointed out an increase in the reactivity of HUA to 5-HT and Hist due to OMC exposure. These alterations in reactivity may be precursors of preeclampsia development and/or gestational hypertension.

Metformin is the most used oral anti-diabetic drug in the world and consequently is commonly found in the aquatic environment. Some studies demonstrated that metformin may act as an endocrine-disrupting-chemical (EDC) in fish, although it does not have a classic EDC structure. In this sense, the aim of this work was to evaluate the potential disrupting effect of metformin in the cardiovascular system through in vitro, ex vivo, and in silico studies. For this purpose, human umbilical artery (HUA) and rat aorta artery (RAA) were used. The toxic concentrations of metformin were determined by a cytotoxicity assay and in silico simulations were performed to analyze the interactions of metformin with hormonal receptors. Our results show that metformin decreases viability of the smooth muscle cells. Moreover, metformin induces a vasorelaxant effect in rat aorta and human models by an endothelium-dependent and -independent pathways. Furthermore, docking simulations showed that metformin binds to androgen receptors (AR) and estrogen receptors (ERα and ERβ). In conclusion, the in silico assays suggested that metformin has the potential to be an endocrine disruptor, acting mainly on ERα. Further studies are needed to use metformin in pregnant women without impairing the cardiovascular health of the future generation.

We would like to thank the HBM4EU team including Petra Apel (UBA), Matthieu Meslin and Christophe Rousselle (Anses) for their scientific contribution, as well as the ANSES Working Group on endocrine disruption, and its scientific Anses coordinators François Pouzaud and Sandrine Charles.; International audience; The close structural analogy of bisphenol (BP) S with BPA, a recognized endocrine-disrupting chemical and a substance of very high concern in the European Union, highlights the need to assess the extent of similarities between the two compounds and carefully scrutinize BPS potential toxicity for human health. This analysis aimed to investigate human health toxicity data regarding BPS, to find a point of departure for the derivation of human guidance values. A systematic and transparent methodology was applied to determine whether European or international reference values have been established for BPS. In the absence of such values, the scientific literature on human health effects was evaluated by focusing on human epidemiological and animal experimental studies. The results were analyzed by target organ/system: male and female reproduction, mammary gland, neurobehavior, and metabolism/obesity. Academic experimental studies were analyzed and compared to regulatory data including subchronic studies and an extended one-generation and reproduction study. In contrast to the regulatory studies, which were performed at dose levels in the mg/kg bw/day range, the academic dataset on specific target organs or systems showed adverse effects for BPS at much lower doses (0.5-10 μg/kg bw/day). A large disparity between the lowest-observed-adverse-effect levels (LOAELs) derived from regulatory and academic studies was observed for BPS, as for BPA. Toxicokinetic data on BPS from animal and human studies were also analyzed and showed a 100-fold higher oral bioavailability compared to BPA in a pig model. The similarities and differences between the two bisphenols, in particular the higher bioavailability of BPS in its active (non-conjugated) form and its potential impact on human health, are discussed. Based on the available experimental data, and for a better human protection, we propose to derive human reference values for exposure to BPS from the N(L)OAELs determined in academic studies.

Background and Objectives: Fetal growth abnormalities increase the risk of negative perinatal and long-term outcomes. Bisphenol A (BPA) is a ubiquitous endocrine-disrupting chemical to which humans may be exposed in a number of ways, such as from the environment, via various consumer products, and through the individual’s diet. Since the compound possesses estrogen-mimicking properties and exerts epigenetic and genotoxic effects, it has been associated with harmful effects impacting the entire spectrum of human life, including, vitally, the intrauterine period. We investigated the role of maternal exposure to BPA in abnormal fetal growth velocity, both impaired and excessive. Materials and Methods: Amniotic fluid samples were collected from 35 women who underwent amniocentesis early in the second trimester due to medical reasons. Pregnancies were followed until delivery, and birth weights were recorded. The amniotic fluid samples were subsequently divided into three groups based on fetal birth weight, as follows: AGA (appropriate for gestational age), SGA (small for gestational age), and LGA (large for gestational age). Amniotic fluid BPA levels were determined by gas chromatography coupled with mass spectrometry. Results: BPA was detected in 80% (28/35) of our amniotic fluid samples. Median concentration was 281.495 pg/mL and ranged from 108.82 pg/mL to 1605.36 pg/mL. No significant association was observed between the study groups regarding BPA concentration. A significant positive correlation between amniotic fluid BPA concentration and birth weight centile (r = 0.351, p-value = 0.039) was identified. BPA levels were also inversely associated with gestational age in pregnancies at term (between 37 and 41 weeks) (r = −0.365, p-value = 0.031). Conclusions: Our findings suggest that maternal exposure to BPA during the early second trimester of pregnancy can potentially contribute to increased birthweight percentiles and to decreased gestational age in pregnancies at term.

Prenatal exposure to metabolism-disrupting chemicals (MDCs) has been linked to birth weight, but the molecular mechanisms remain largely unknown. In this study, we investigated gene expressions and biological pathways underlying the associations between MDCs and birth weight, using microarray transcriptomics, in a Belgian birth cohort. Whole cord blood measurements of dichlorodiphenyldichloroethylene (p,p’-DDE), polychlorinated biphenyls 153 (PCB-153), perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and transcriptome profiling were conducted in 192 mother–child pairs. A workflow including a transcriptome-wide association study, pathway enrichment analysis with a meet-in-the-middle approach, and mediation analysis was performed to characterize the biological pathways and intermediate gene expressions of the MDC–birth weight relationship. Among 26,170 transcriptomic features, we successfully annotated five overlapping metabolism-related gene expressions associated with both an MDC and birth weight, comprising BCAT2, IVD, SLC25a16, HAS3, and MBOAT2. We found 11 overlapping pathways, and they are mostly related to genetic information processing. We found no evidence of any significant mediating effect. In conclusion, this exploratory study provides insights into transcriptome perturbations that may be involved in MDC-induced altered birth weight.

Objective: The use of endocrine-disrupting chemicals (EDCs) such as bisphenol A (BPA) in plastics manufacturing, agriculture, livestock, and paint manufacturing increas daily. The water treated in wastewater treatment plants is used in many areas such as irrigation of parks and gardens, and reinforcement of underground water resources. However, whether the treatment process eliminates EDCs in wastewater is not exactly known, and determining this as well as the amounts of these chemicals in treated water are important in terms of protecting the environment and human health. The aim of the study was to determine BPA and phthalate concentrations in the influent and effluent flow samples obtained from wastewater treatment plants. Materials and Methods: BPA and phthalate concentrations were measured in influent and effluent flow samples using the enzyme- linked immunosorbent assay (ELISA) method. BPA and phthalate measurements were performed as competitive measurements of BPA and total phthalates in samples using specific monoclonal antibodies. Results: BPA and phthalate levels were measured respectively as 7.69 μg/L and 78.27 μg/L in the influent water samples and 3.17 μg/L and 25.56 μg/L in the effluent water samples. The concentration of BPA and phthalates in the effluent samples decreased significantly compared to the influent water samples. Conclusion: This study is believed to shed light on the importance of monitoring BPA and phthalate concentrations in wastewater treatment plants and inspections for detecting other EDCs in wastewater.

There is growing concern regarding the health and safety issues of endocrine-disrupting chemicals (EDCs). Long-term exposure to EDCs has serious adverse health effects through both hormone-direct and hormone-indirect ways. Accordingly, some EDCs can be a pathogen and an inducer to the susceptibility of disease, even if they have a very low affinity on the estrogen receptor, or no estrogenic effect. Endoplasmic reticulum (ER) stress recently attracted attention in this research area. Because ER and ER stress could be key regulators of the EDC’s adverse effects, such as the malfunction of the organ, as well as the death, apoptosis, and proliferation of a cell. In this review, we focused on finding evidence which shows that EDCs could be a trigger for ER stress and provide specific examples of EDCs, which are known to cause ER stress currently.

Bisphenol A (BPA) is an environmental risk factor for autism spectrum disorder (ASD). BPA exposure dysregulates ASD-related genes in the hippocampus and neurological functions of offspring. However, whether prenatal BPA exposure has an impact on genes in the prefrontal cortex, another brain region highly implicated in ASD, and through what mechanisms have not been investigated. Here, we demonstrated that prenatal BPA exposure disrupts the transcriptome–interactome profiles of the prefrontal cortex of neonatal rats. Interestingly, the list of BPA-responsive genes was significantly enriched with known ASD candidate genes, as well as genes that were dysregulated in the postmortem brain tissues of ASD cases from multiple independent studies. Moreover, several differentially expressed genes in the offspring’s prefrontal cortex were the targets of ASD-related transcription factors, including AR, ESR1, and RORA. The hypergeometric distribution analysis revealed that BPA may regulate the expression of such genes through these transcription factors in a sex-dependent manner. The molecular docking analysis of BPA and ASD-related transcription factors revealed novel potential targets of BPA, including RORA, SOX5, TCF4, and YY1. Our findings indicated that prenatal BPA exposure disrupts ASD-related genes in the offspring’s prefrontal cortex and may increase the risk of ASD through sex-dependent molecular mechanisms, which should be investigated further.

Anaerobic digestion of biomass has increasing implementation for bioenergy production. The solid by-product of this technology, i.e., the digestate, has relevant potential in agricultural and environmental applications. This study explored the capacity of a digestate from mixed feedstock to remove from water four endocrine-disrupting chemicals, namely the pesticides metribuzin (MET) and boscalid (BOS) and the xenoestrogens bisphenol A (BPA) and 4-tert-octylphenol (OP). The surface micromorphology and functional groups of the digestate were investigated using scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR) spectroscopy, respectively. Results of sorption kinetics showed that all compounds reached the steady state in a few hours according to a pseudo-first-order model in the cases of MET and OP, a pseudo-second-order model for BOS and both models in the case of BPA. Data of adsorption isotherms were fitted to the Henry, Freundlich, Langmuir and Temkin equations. The adsorption of MET preferentially followed the non-linear Freundlich model, whereas the adsorption of the other compounds was properly described by both the linear and Freundlich models. The organic carbon partition coefficients, KOC, were 170, 1066, 256 and 2180 L kg−1 for MET, BOS, BPA and OP, respectively. The desorption of BOS, BPA and OP was slow and incomplete, indicating a phenomenon of hysteresis. In conclusion, the digestate showed a remarkable efficiency in the removal of the compounds, especially those with high hydrophobicity, thus behaving as a promising biosorbent for environmental remediation.

Cyclic siloxane octamethylcyclotetrasiloxane (D4) is an endocrine-disrupting chemical (EDC) used widely in detergent products, cosmetics, and personal care products. Recently, robust toxicological data for D4 has been reported, but the adverse effects of D4 on brain development are unknown. Here, pregnant mice on a gestational day 9.5 were treated daily with D4 to postnatal day 28, and the offspring mice were studied. The prenatal D4-treated mice exhibited cognitive dysfunction, limited memory, and motor learning defect. Moreover, prenatal D4 exposure reduced the proliferation of neuronal progenitors in the offspring mouse brain. Next, the mechanism(s) through which D4 regulated the cell cycle was investigated. Aberrant gene expression, such as cyclin-dependent kinases CDK6 and cyclin-dependent kinase inhibitor p27, was found in the prenatal D4-treated mice. Furthermore, estrogen receptor ERa and ERb increased in the brain of prenatal D4-treated mice. Overall, these findings suggest that D4 exerts estrogen activity that affects the cell cycle progression of neuronal progenitor cells during neurodevelopment, which may be associated with cognitive deficits in offspring.

Endocrine-disrupting chemicals (EDCs) are different natural and synthetic chemicals that may interfere with several mechanisms of the endocrine system producing adverse developmental, metabolic, reproductive, and neurological effects in both human beings and wildlife. Among pesticides, numerous chemicals have been identified as EDCs. MicroRNAs (miRNAs) can regulate gene expression, making fine adjustments in mRNA abundance and regulating proteostasis. We hypothesized that exposure to low doses of atrazine, cypermethrin, and vinclozolin may lead to effects on miRNA expression in SH-SY5Y cells. In particular, the exposure of SH-SY5Y cells to subtoxic concentrations of vinclozolin is able to downregulate miR-29b-3p expression leading to the increase in the related gene expression of ADAM12 and CDK6, which may promote a pro-oncogenic response through the activation of the PI3K/Akt/mTOR pathway and counteracting p53 activity. A better understanding of the molecular mechanisms of EDCs could provide important insight into their role in human disease.

Diethylstilbestrol (DES), a transplacental endocrine-disrupting chemical, was prescribed to pregnant women for several decades. The number of women who took DES is hard to know precisely, but it has been estimated that over 10 million people have been exposed around the world. DES was classified in the year 2000 as carcinogenic to humans. The deleterious effects induced by DES are very extensive, such as abnormalities or cancers of the genital tract and breast, neurodevelopmental alterations, problems associated with socio-sexual behavior, and immune, pancreatic and cardiovascular disorders. Not only pregnant women but also their children and grandchildren have been affected. Epigenetic alterations have been detected, and intergenerational effects have been observed. More cohort follow-up studies are needed to establish if DES effects are transgenerational. Even though DES is not currently in use, its effects are still present, and families previously exposed and their later generations deserve the continuity of the research studies.

Bisphenol A is an extremely high-volume chemical widely used in polycarbonate plastics, the linings of food and beverage tins, and shopping receipts. Canadians are ubiquitously exposed to bisphenol A and research shows that exposure at environmentally relevant doses causes endocrine disruption. Recent risk assessments and exposure estimates by the European Food Safety Authority have guided increased restrictions around the use of bisphenol A and established a lower tolerable daily intake, while the CLARITY-BPA program in the United States identified several adverse effects below this exposure level. Within the context of bisphenol toxicity and international regulation, this paper describes the need for revised bisphenol A risk assessments in Canada. Completed in 2008, the most recent bisphenol A risk assessment conducted by Health Canada does not include risks from alternative bisphenols or non-dietary exposure. It also does not account for the additive effects caused by simultaneous exposure to multiple endocrine-disrupting chemicals.

Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical (EDC), is increasingly hypothesized to be a factor contributing to changes in fetal growth velocity. BPA exposure may be environmental, occupational, and/or dietary, with canned foods and plastic bottles contributing significantly. Our systematic review aims to evaluate the current literature and to investigate the role of BPA in abnormal fetal growth patterns. A search was conducted in the PubMed and Cochrane databases. A total of 25 articles met the eligibility criteria and were included in this systematic review. Eleven of them failed to show a clear relationship between BPA and abnormal fetal growth. The majority of the remaining studies (9/14) found an inverse association of BPA with indicators of fetal growth, whereas three studies suggested increased fetal growth, and two studies produced contradictory findings. Of note, both of the studies that collected a sample (amniotic fluid) directly reflecting BPA concentration in the fetus during the first half of pregnancy revealed an inverse association with birth weight. In conclusion, there is mounting evidence that combined exposure to BPA from dietary and non-dietary sources during pregnancy may contribute to abnormal fetal growth; a tendency towards fetal growth restriction was shown, especially when exposure occurs during the first half.

According to the available data, environmental pollution is a serious problem all over the world. Between 2015 and 2016, pollution was responsible for approximately nine million deaths worldwide. They also include endocrine disrupting chemicals (EDCs) that can interfere with the functioning of the thyroid gland. They are characterized by high persistence in the environment. These substances can enter the body through the gastrointestinal tract, respiratory system, as well as contact with the skin and overcome the placental barrier. EDC can be found in food, water, and personal care products. They can get into food from the environment and as a result of their migration to food products and cosmetics from packaging. EDCs can disrupt the functioning of the thyroid gland through a number of mechanisms, including disrupting the activation of thyroid receptors and the expression of genes that are related to the metabolism, synthesis, and transport of thyroid hormones (HT). There is a need to strengthen the food safety policy that aimed at the use of appropriate materials in direct contact with food. At the same time, an important action is to reduce the production of all waste and, when possible, use biodegradable packaging, which may contribute to the improvement of the quality of the entire ecosystem and the health of food, thus reducing the risk of developing thyroid diseases.

Obesity is now a worldwide pandemic. The usual explanation given for the prevalence of obesity is that it results from consumption of a calorie dense diet coupled with physical inactivity. However, this model inadequately explains rising obesity in adults and in children over the past few decades, indicating that other factors must be important contributors. An endocrine-disrupting chemical (EDC) is an exogenous chemical, or mixture that interferes with any aspect of hormone action. EDCs have become pervasive in our environment, allowing humans to be exposed daily through ingestion, inhalation, and direct dermal contact. Exposure to EDCs has been causally linked with obesity in model organisms and associated with obesity occurrence in humans. Obesogens promote adipogenesis and obesity, in vivo, by a variety of mechanisms. The environmental obesogen model holds that exposure to obesogens elicits a predisposition to obesity and that such exposures may be an important yet overlooked factor in the obesity pandemic. Effects produced by EDCs and obesogen exposure may be passed to subsequent, unexposed generations. This “generational toxicology” is not currently factored into risk assessment by regulators but may be another important factor in the obesity pandemic as well as in the worldwide increases in the incidence of noncommunicable diseases that plague populations everywhere. This review addresses the current evidence on how obesogens affect body mass, discusses long-known chemicals that have been more recently identified as obesogens, and how the accumulated knowledge can help identify EDCs hazards.

The incidence of obesity has dramatically increased over the last decades. Recently, there has been a growing interest in the possible association between the pandemics of obesity and some endocrine-disrupting chemicals (EDCs), termed “obesogens”. These are a heterogeneous group of exogenous compounds that can interfere in the endocrine regulation of energy metabolism and adipose tissue structure. Oral intake, inhalation, and dermal absorption represent the major sources of human exposure to these EDCs. Recently, epigenetic changes such as the methylation of cytosine residues on DNA, post-translational modification of histones, and microRNA expression have been considered to act as an intermediary between deleterious effects of EDCs and obesity development in susceptible individuals. Specifically, EDCs exposure during early-life development can detrimentally affect individuals via inducing epigenetic modifications that can permanently change the epigenome in the germline, enabling changes to be transmitted to the next generations and predisposing them to a multitude of diseases. The purpose of this review is to analyze the epigenetic alterations putatively induced by chemical exposures and their ability to interfere with the control of energy metabolism and adipose tissue regulation, resulting in imbalances in the control of body weight, which can lead to obesity.

Bisphenol A (BPA), an endocrine-disrupting chemical, is present in everyday-used consumables and common household products. Although the side effects of BPA have been sufficiently explored, little is known the effects of environmentally relevant low levels of BPA on chondrogenesis in skeletal development. Here we used a morphological approach to investigate whether exposure to BPA (0, 0.0038, 0.05, 0.1, 1.0 μM) could affect craniofacial cartilage development of zebrafish embryo. Furthermore, we sought to determine receptor-mediated BPA induced chondrogenesis toxicity by co-exposing developing embryos to BPA and various inhibitors. Low-dose BPA affected heart rate and induced body and head elongation of larvae. Quantitative morphometric and histopathological analysis revealed that BPA exposure changed the angle and length of craniofacial cartilage elements and disrupted chondrocytes. BPA induced pharyngeal cartilage defects via multiple cellular pathways, including estrogen receptor, androgen receptor, and estrogen-related receptors. Our findings demonstrate that BPA alters the normal development of cartilage and craniofacial structures in zebrafish embryos. Furthermore, in this study we find multiple cellular pathways mediating the effects of BPA-induced craniofacial chondrogenesis toxicity. Further experiments will allow for establishing a connection between BPA and increased risk of congenital malformation of the facial cranium in BPA-exposed populations.

A quick and inexpensive validated method, based on sample treatment by liquid-liquid microextraction followed by liquid chromatography (LC) coupled with ultraviolet tandem fluorescence detection is proposed for the determination of 15 multiclass pollutants both in serum and in saliva, as a simple and easy to draw matrix. The method was set up and validated according to European guidelines. The compounds of interest include some endocrine-disrupting chemicals (i.e. bisphenol A, bisphenol B, bisphenol E, bisphenol F, bisphenol AF, bisphenol A diglycidyl ether, bisphenol M, diethylhexyl phthalate, monoethylhexyl phthalate, triclosan and 4-nonylphenol), as well as other pollutants belonging to the class of volatile organic compounds (2-chlorophenol, 1,2 dichlorobenzene, 1,2,4,5-tetrachlorobenzene). The limits of quantifications ranged from 2.28 × 10-3 μg mL-1 (bisphenol A diglycidyl ether) to 6.29 μg mL-1 (diethylhexyl phthalate), while those of detection ranged from 0.068 × 10-3 μg mL-1 (bisphenol A diglycidyl ether) to 1.031 μg mL-1 (diethylhexyl phthalate). To test method suitability, it was applied to real saliva and serum samples of healthy human volunteers and was found to meet the demands of the laboratories handling simple and relatively inexpensive equipment for screening oriented at rapid and reliable contamination assessment of a population.

Introduction: Indomethacin (NSAID) is an endocrine-disrupting chemical (EDC) that has been demonstrated to induce alterations in reproductive organs while Black Tea Extract (BTE), an antioxidant, is capable of protecting against EDC-induced alterations. Aims & Objectives: The current study was designed to investigate the role of black tea extract on biochemical, hormonal, and histological changes in the prostate of male Wister rats treated with indomethacin drugs. Materials and Methods: Adult rats were divided into four groups (n = 6/group); Group I (control); Group II (indomethacin, 5 mg/kg b.wt.; i.p.); Group III (BTE, 2.5 g tea leaf/dL of water, i.e., 2.5% of aqueous BTE, orally) and Group IV (indomethacin + BTE) for 21 successive days. Sperm Count, Motility, Prostate-Specific Antigen (PSA), Testosterone levels, total protein, GSH, CAT, and MDA levels and the histopathological aspects of the prostate gland were studied. Statistical Analysis: Statistical comparisons were performed using the one-way ANOVA, followed by post-hoc t-test. Results: Indomethacin treated rats “Group” II exhibited a significant (P≤0.05) declining in the sperm count, motility PSA and testosterone concentration, total protein, GSH concentration, and CAT activity. A high concentration of MDA levels in the serum indicated the appearance of oxidative stress status in the Wister rats. The indomethacin had a significant impact on the weight, and histopathology of glandular “acini” and excretory “ducts” revealed hyperplasia, intra-luminal budding of epithelia as well as tubular atrophy. Conclusion: Administration of black tea extract abrogated the toxic effect induced by indomethacin on the prostate gland.

Growing evidence suggests the causal link between the endocrine-disrupting chemicals (EDCs) and the global obesity epidemics, in the context in the so-called "obesogenic environment". Dietary intake of contaminated foods and water, especially in association with unhealthy eating pattern, and inhalation of airborne pollutants represent the major sources of human exposure to EDCs. This is of particular concern in view of the potential impact of obesity on chronic non-transmissible diseases, such as type 2 diabetes, cardiovascular disease, and hormone-sensitive cancers. The key concept is the identification of adipose tissue not only as a preferential site of storage of EDCs, but also as an endocrine organ and, as such, susceptible to endocrine disruption. The timing of exposure to EDCs is critical to the outcome of that exposure, with early lifetime exposures (e.g., fetal or early postnatal) particularly detrimental because of their permanent effects on obesity later in life. Despite that the mechanisms operating in EDCs effects might vary enormously, this minireview is aimed to provide a general overview on the possible association between the pandemics of obesity and EDCs, briefly describing the endocrine mechanisms linking EDCs exposure and latent onset of obesity.

Prenatal exposure to bisphenol A (BPA), one of the most prevalent endocrine disrupting chemicals, is associated with lung dysfunction and diseases in later life. However, it is unknown if this association has a fetal origin. In this thesis, a series of in vivo and in vitro experiments were conducted to determine the effects of prenatal exposure to BPA on fetal lung development, and define the underlying molecular mechanisms. Environmentally relevant doses of BPA were administered to pregnant mice via diet from embryonic day (E) 7.5 to 18.5. Fetal lungs were analyzed at E18.5 for changes in structure and expression of key molecular markers of lung maturation. My main findings were: (a) BPA severely retards fetal lung maturation as evidenced by diminished alveolar airspace and thickened septa, hallmarks of lung immaturity; (b) this immaturity is characterized by aberrant alveolar epithelial type I cell differentiation; and (c) the effects of BPA are likely mediated through the glucocorticoid signaling pathway, because the expression of epithelial sodium channel g (ENaCγ), a well-known glucocorticoid receptor (GR) target gene, is down-regulated in BPA-exposed fetal lungs. Moreover, maternal administration of dexamethasone rescues the lung immaturity phenotype. However, the precise molecular mechanisms by which BPA represses ENaCγ in lung cells were unknown. This important question was addressed using the A549 human lung epithelial cell line as an in vitro model system. I found that that (d) both dexamethasone and siRNA-mediated knockdown of GR expression blocked the inhibitory effects of BPA on ENaCγ expression, indicating that BPA suppresses ENaCγ via inhibition of GR activity. Given that BPA is known to function as a pro-inflammatory factor via the estrogen receptor b (ER b), and a mutual antagonism exists between the pro-inflammatory transcriptional factor NF-κB and GR, I then explored and provide evidence supporting the notion that (e) BPA acts on ER-β to activate the NF-κB signaling pathway, which in turn leads to diminished GR activity and consequent repression of ENaCγ expression in lung cells. Taken together, these findings demonstrate that prenatal exposure to BPA disrupts fetal lung maturation, and suggest a fetal origin for BPA-induced lung dysfunction and diseases.

Background: The organochlorine dichlorodiphenyltrichloroethane (DDT), a known estrogen mimic and endocrine disruptor, has been linked to animal and human disorders. However, the detailed mechanism(s) by which DDT affects cellular physiology remains incompletely defined. Objectives: We and others have shown that DDT activates cell-signaling cascades, culminating in the activation of estrogen receptor-dependent and -independent gene expression. Here, we identify a mechanism by which DDT alters cellular signaling and gene expression, independent of the estrogen receptor. Methods: We performed quantitative polymerase chain reaction array analysis of gene expression in MCF-7 breast cancer cells using either estradiol (E2) or o,p´-DDT to identify distinct cellular gene expression responses. To elucidate the mechanisms by which DDT regulates cell signaling, we used molecular and pharmacological techniques. Results: E2 and DDT treatment both altered the expression of many of the genes assayed, but up-regulation of vascular endothelial growth factor A (VEGFA) was observed only after DDT treatment, and this increase was not affected by the pure estrogen receptor α antagonist ICI 182780. Furthermore, DDT increased activation of the HIF-1 response element (HRE), a known enhancer of the VEGFA gene. This DDT-mediated increase in HRE activity was augmented by the coactivator CBP (CREB-binding protein) and was dependent on the p38 pathway. Conclusions: DDT up-regulated the expression of several genes in MCF-7 breast cancer cells that were not altered by treatment with E2, including VEGFA. We propose that this DDT-initiated, ER-independent stimulation of gene expression is due to DDT’s ability to initiate crosstalk between MAPK (mitogen-activated protein kinase) signaling pathways and transcriptional coactivators.

The prevalence of allergic diseases has increased in recent decades. Allergic diseases, particularly asthma, are complex diseases with strong gene-environment interactions. Epidemiological studies have identified a variety of risk factors for the development of allergic diseases. Among them, endocrine-disrupting chemicals (EDCs) play an important role in triggering or exacerbating these diseases. 4-Nonylphenol (NP) and 4-octylphenol (OP)—two major alkylphenols—have been recognized as common toxic and xenobiotic endocrine disrupters. Due to their low solubility, high hydrophobicity, and low estrogenic activity, they tend to accumulate in the human body and may be associated with the adverse effects of allergic diseases. Recently, new evidence has supported the importance of alkylphenols in the in vitro allergic response. This review focuses on the effects of alkylphenols on several key cell types in the context of allergic inflammation.

During recent decades more than 100,000 new chemicals have been introduced as common consumer products into our environment. Among these chemicals, endocrine-disrupting chemicals (EDCs) are of particular concern owing to their toxicity in animal studies and their impacts on human health. EDCs are ubiquitous in the environment, including the air, water, and soil. The endocrine-disrupting effect of EDCs has been found to imitate the action of steroid hormones and promote several endocrine and reproductive disorders in both animal and human studies. In the present review, we focus on the effects of EDCs on the immune system. EDCs interfere with the synthesis of cytokines, immunoglobulins, and inflammatory mediators, and they also affect the activation and survival of immune cells. The dysfunction of the immune system caused by EDCs may lead to the attenuation of immunity (immunodeficiency) against infection or hyperreactivity of immune responses (allergy and autoimmune disease). In this review, we summarize epidemiologic, animal, and cell studies to demonstrate the potential effects of EDCs on immunity, allergy, and autoimmune diseases. We also address the impact of EDCs on epigenetic regulation.

Background: Chemicals having estrogenic activity (EA) reportedly cause many adverse health effects, especially at low (picomolar to nanomolar) doses in fetal and juvenile mammals. Objectives: We sought to determine whether commercially available plastic resins and products, including baby bottles and other products advertised as bisphenol A (BPA) free, release chemicals having EA. Methods: We used a roboticized MCF-7 cell proliferation assay, which is very sensitive, accurate, and repeatable, to quantify the EA of chemicals leached into saline or ethanol extracts of many types of commercially available plastic materials, some exposed to common-use stresses (microwaving, ultraviolet radiation, and/or autoclaving). Results: Almost all commercially available plastic products we sampled—independent of the type of resin, product, or retail source—leached chemicals having reliably detectable EA, including those advertised as BPA free. In some cases, BPA-free products released chemicals having more EA than did BPA-containing products. Conclusions: Many plastic products are mischaracterized as being EA free if extracted with only one solvent and not exposed to common-use stresses. However, we can identify existing compounds, or have developed, monomers, additives, or processing agents that have no detectable EA and have similar costs. Hence, our data suggest that EA-free plastic products exposed to common-use stresses and extracted by saline and ethanol solvents could be cost-effectively made on a commercial scale and thereby eliminate a potential health risk posed by most currently available plastic products that leach chemicals having EA into food products.

In view of the heath problems induced by the presence into the environment of endocrine disruptors, laccase from Trametes versicolor was covalently immobilized on a chemically modified polypropylene membrane in order to remove phenol and its derivatives from polluted waters. Using phenol as substrate model the optimal immobilization conditions were determined. The immobilized laccase exhibited maximal enzyme activity at pH 5.5 and optimal temperature at 55 °C. These operative parameters have been compared with those obtained with the soluble laccase in order to ascertain the immobilization effect. When employed in a bioreactor operating under isothermal conditions the immobilized laccase was able to oxidize a wide range of phenolic substrates. In particular it was found that some phenol derivatives (2-CP, 3-CP, 4-CP, NP and chlorophene) were oxidized at a similar rate than phenol, other derivatives (paracetamol, 3-MP and chloroxyphenol) at a smaller rate, while others (2,4-DCP and BPA) at higher rate. When the catalytic membrane was employed in a non-isothermal reactor the reaction rate increased with the increase of the applied temperature difference. Practically the increase of the laccase oxidative power under the non-isothermal conditions followed the same sequence observed under isothermal conditions. Interesting enough, the percentage increase of enzyme reaction rate under non-isothermal conditions resulted higher in the cases in which the isothermal reaction rate was smaller. When the reduction of the production times by the presence of a temperature gradient is considered, the measured values strongly candidate the technology of non-isothermal bioreactors as a useful tool in processes of detoxification of waste waters polluted by endocrine disruptors of phenolic origin. © 2010 Elsevier B.V. All rights reserved.

This paper explores the degradation of a model pollutant, bisphenol A, by an advanced oxidation process that combines sonolysis, Fe2+, and TiO2 in a photoassisted process. Experiments were done under saturated oxygen conditions. The effect of different Fe2+ (0.56 and 5.6 mg/L) and TiO2 (10 and 50 mg/L) concentrations was investigated on both the elimination and mineralization of the pollutant. A pronounced synergistic effect that led to the complete and rapid elimination of dissolved organic carbon (DOC) was observed even at low catalyst loadings. In this system, almost a complete removal of DOC (93%) was observed after 4 h using 10 and 5.6 mg/L of TiO2 and Fe2+, respectively, whereas at the same time, only 5, 6, and 22% of DOC was removed by an individual process alone (TiO2 photocatalysis, ultrasound, and photo-Fenton, respectively). In this system, ultrasound has the principal role of eliminating the initial substrate and providing hydrogen peroxide for the photocatalytic systems, while photo-Fenton and TiO2 photocatalysis are mainly responsible for the transformation of the intermediates in CO2 and H2O. The role of H2O2 generated from the sonochemical process is also discussed. (C) 2010 Elsevier Ltd. All rights reserved.

Background Exposure to environmental endocrine-disrupting chemicals (EDCs) is often associated with dysregulated immune homeostasis, but the mechanisms of action remain unclear. Objectives The aim of this study was to test a hypothesis that EDCs regulate the functions of human dendritic cells, a front-line, immunoregulatory cell type in contact with the environment. Methods We investigated circulating myeloid dendritic cells (mDCs) from five subjects and measured their responses, with or without coculture with autologous T cells, to two common EDCs, nonylphenol (NP) and 4-octylphenol (4-OP). EDC-associated cytokine responses, signaling events, and histone modifications were examined using ELISA, Western blotting, and chromatin immunoprecipitation (ChIP) assays, respectively. Results In all cases, mDCs treated with NP or 4-OP demonstrated increased expression of tumor necrosis factor-α (TNF-α) but decreased baseline and lipopolysaccharide (LPS)-induced (interleukin) (IL)-10 production; the increase in TNF-α was partially reversible by an estrogen receptor (ER) antagonist. Activation of the MKK3/6-p38 signaling pathway marked the effect of NP on TNF-α expression, concomitant with enhanced levels of methyltranferase complex [mixed-lineage leukemia (MLL) and tryptophan-aspartic acid repeat domain 5 (WDR5)] in the nucleus and of trimethylated H3K4, acetylated H3, and H4 at the TNFA gene locus. Further, up-regulated TNF-α expression was significantly suppressed in NP-treated mDCs by a histone acetyltransferase inhibitor. In the presence of NP-treated mDCs, T cells showed increased levels of IL-13 but decreased expression of interferon-γ. Conclusions These results suggest that NP and 4-OP may have functional effects on the response of mDCs via, in part, the ER, MKK3/6-p38 MAPK signaling pathway, and histone modifications, with subsequent influence on the T-cell cytokine responses.

In the last 10 years, good evidence has become available to show that the combined effects of endocrine disruptors (EDs) belonging to the same category (e.g., estrogenic, antiandrogenic, or thyroid- disrupting agents) can be predicted by using dose addition. This is true for a variety of end points representing a wide range of organizational levels and biological complexity. Combinations of EDs are able to produce significant effect, even when each chemical is present at low doses that individually do not induce observable effects. However, comparatively little is known about mixtures composed of chemicals from different classes of EDs. Nevertheless, I argue that the accumulated evidence seriously undermines continuation with the customary chemical-by-chemical approach to risk assessment for EDs. Instead, we should seriously consider group-wise regulation of classes of EDs. Great care should be taken to define such classes by using suitable similarity criteria. Criteria should focus on common effects, rather than common mechanisms. In this review I also highlight research needs and identify the lack of information about exposure scenarios as a knowledge gap that seriously hampers progress with ED risk assessment. Future research should focus on investigating the effects of combinations of EDs from different categories, with considerable emphasis on elucidating mechanisms. This strategy may lead to better-defined criteria for grouping EDs for regulatory purposes. Also, steps should be taken to develop dedicated mixtures exposure assessment for EDs.

Phytoestrogens and organochlorine pesticides in the diet of laboratory animals are a possible source of interference in bioassays that assess estrogenic activity. In the present study, we investigated the levels of dietary phytoestrogens, organochlorine pesticides and the estrogenic activity of various diets for an experimental fish and discuss the potential contribution of these substances to estrogenic activity, in comparison with those used in previous studies. After hydrolysis with β-glucuronidase, genistein and daidzein were detected in all of the diets, and there were no significant differences in the contents of these substances among present and previous investigations. In addition, organochlorine pesticides, such as hexachlorobenzene (HCB), β-benzene hexachloride (β-BHC), γ-BHC, trans-nonachlor, and/or endrin, were detected in most fish diets. All of these diets exhibited higher levels of activation of β estrogen receptors than with α estrogen receptors in an in vitro yeast-based bioassay. These results indicate that phytoestrogens, such as genistein and daidzein, were the main substances contributing to the estrogenic activity of the diet. Moreover, some diets may exert estrogenic activity in in vivo tests, indicating the necessity for more careful selection of the feeding diet and measurement of estrogenic substances when performing routine screening assays for endocrine-disrupting chemicals., Journal of Health Science v.51(2) p.212-219, 2005

Two kinds of absorbents (1 and 2) for endocrine-disrupting chemicals have been synthesized from β-cyclodextrin, in which 1 is β-cyclodextrin polymer combined with rice husks ash of 112 m2/g specific surface and 2 is β-cyclodextrin polyalkylene polyamine linked with polystyrene. Those absorbents are not soluble in water and exhibited removal capability for endocrine disruptor in aqueous layer with high efficiency. Absorbent 1 as well 2 show high selectivity for 4, 4-OE-isopyropidendiphenol (bisphenol A) with 97.15% and 98% removal rate respectively. On the other hand, β-cyclodextrin polymer, which is non-coexistent with rice husks ash and polystyrene itself show 63 % and only 2% removal rate of bisphenol A respectively. It is useful in a combination between β-cyclodextrin and rice husks ash or polystyrene, however, the complex of β-cyclodextrin polymer combined with amorphous silica (5) is not useful, which exhibits less 60% removal rate of bisphenol A.

This article presents an overview of the analytical methods for the determination of target endocrine-disrupting chemicals (EDCs) in airborne particles. Among EDCs, we selected four important groups of compounds (alkylphenols, bisphenol A, phthalates, and natural and synthetic sex hormones) because of their worldwide production, environmental ubiquity and toxicity. Due to their physical and chemical properties (high molecular weight, low/medium polarity and volatility), these EDCs can be present in the atmosphere mainly associated with particulate matter. EDCs linked to small particles (PM10 and PM2.5) can penetrate into the respiratory system and damage human health. We discuss aspects of analytical methodology, such as sampling, extraction, and determination. We review levels found in outdoor and indoor samples. According to the results reported, human exposure to these compounds, especially in indoor environments, should be investigated in more depth, since some compounds, such as dibutyl phthalate and diethyl phthalate, reach levels of μg/m3., Applied Analytical Chemistry Research Group (QANAP), University Institute of Research in Environmental Studies, Department of Analytical Chemistry, University of A Coruña, Campus da Zapateira, Rua da Fraga 10, A Coruña, Spain Water and Soil Quality Research Group, Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Jordi Girona 18-26, Barcelona, Spain Catalan Institute for Water Research (ICRA), Universitat de Girona, Emili Grahit, Parc Científic i Tecnològic, 101, Edifici H2O, Girona, Spain

Vitellogenin (VTG) is believed to be an effective and sensitive biomarker to detect the effects of endocrine-disrupting chemicals (EDCs) on fish. An enzymelinked immunosorbent assay (ELISA) was developed in this study for the quantification of VTG in goldfish (Carassius auratus) blood plasma using a monoclonal antibody against carp lipovitellin which is known to cross-react with goldfish VTG, and a working range of 7.8 to 500 ng VTG/ml was established. A laboratory study involving the dosing of male goldfish for 28 days with a range of bisphenol-A (BPA) concentrations was conducted, and the established ELISA was used. There was no significant induction of VTG at the concentrations of 1 and 10 μg/l. Exposure to 100 μg/l and 1000 μg/l BPA for 28 days significantly elevated plasma VTG concentrations to 201 ± 90 μg/ml and 104552 ± 24920 μg/ml, respectively. To confirm the effect of temperature on VTG induction, male goldfish were exposed to the nominal concentration of 17β-es-tradiol (E2) 100 μg/l at 10° and 30°C for 10 days. Plasma VTG concentration in male goldfish increased more quickly in the fish exposed at 30°C than in those at 10°C. In addition, the plasma VTG concentration in the 30°C group (10463 ± 3268 μg/ml) was higher than that of the 10°C group (1.42 ± 1.6 μg/ml) after 1 day of exposure. Thus goldfish are useful for the investigation of EDCs under various conditions since they are relatively small in size, and the ELISA established in the present study will contribute to various types of research.

Vitellogenin (VTG) is believed to be an effective and sensitive biomarker to detect the effects of endocrine-disrupting chemicals (EDCs) on fish. An enzymelinked immunosorbent assay (ELISA) was developed in this study for the quantification of VTG in goldfish (Carassius auratus) blood plasma using a monoclonal antibody against carp lipovitellin which is known to cross-react with goldfish VTG, and a working range of 7.8 to 500 ng VTG/ml was established. A laboratory study involving the dosing of male goldfish for 28 days with a range of bisphenol-A (BPA) concentrations was conducted, and the established ELISA was used. There was no significant induction of VTG at the concentrations of 1 and 10 μg/l. Exposure to 100 μg/l and 1000 μg/l BPA for 28 days significantly elevated plasma VTG concentrations to 201 ± 90 μg/ml and 104552 ± 24920 μg/ml, respectively. To confirm the effect of temperature on VTG induction, male goldfish were exposed to the nominal concentration of 17β-es-tradiol (E2) 100 μg/l at 10° and 30°C for 10 days. Plasma VTG concentration in male goldfish increased more quickly in the fish exposed at 30°C than in those at 10°C. In addition, the plasma VTG concentration in the 30°C group (10463 ± 3268 μg/ml) was higher than that of the 10°C group (1.42 ± 1.6 μg/ml) after 1 day of exposure. Thus goldfish are useful for the investigation of EDCs under various conditions since they are relatively small in size, and the ELISA established in the present study will contribute to various types of research., Journal of Health Science v.47(2) p.213-218, 2001

Various assays with adult fish have been developed to identify potential endocrine-disrupting chemicals (EDCs) which may cause toxicity via alterations in the hypothalamic-pituitary-gonadal (HPG) axis. These assays can be sensitive and highly diagnostic for key mechanisms such as agonism of the estrogen and androgen receptors (ERs, ARs) and inhibition of steroid synthesis. However, most of the tests do not unambiguously identify AR antagonists. The purpose of this work was to explore the utility of a mixture test design with the fathead minnow (Pimephales promelas) for detecting different classes of EDCs including AR antagonists. Adults of both sexes were exposed via the water to EDCs with diverse mechanisms of action in the absence or presence of 17beta-trenbolone (TB), a potent AR agonist which masculinizes female fathead minnows. Similar to previous studies with the model AR antagonists flutamide and vinclozolin, exposure of females to the AR antagonist cyproterone acetate in the presence of TB decreased expression of an easily-observed masculinization response, nuptial tubercle formation. Mixture studies with TB and the model ER agonists, 17alpha-ethinylestradiol and bisphenol A, also showed inhibition of tubercle formation in the females, but unlike the AR antagonists, the estrogens markedly induced synthesis of vitellogenin (VTG: egg yolk protein), particularly in males. The ER agonists also offset TB-induced depressions in plasma VTG concentrations in female fish. Additional mixture experiments were conducted with TB and triclocarban, an anti-microbial reported to enhance AR-mediated responses, or ammonia, a "negative control" with no known direct effects on HPG function. Neither chemical affected VTG status in males or females in the absence or presence of TB; however, both slightly enhanced TB-induced tubercle formation in females. Based on studies described herein and elsewhere with the fathead minnow, a TB co-exposure assay appears to be an effective approach for clearly identifying AR antagonists as well as potential EDCs with other relevant mechanisms of action.